Abstract This application, ?Development of novel small molecule analgesics modulating the nNOS-NOS1AP protein- protein interaction,? addresses the critical need for more effective medications to treat chronic neuropathic pain affecting ~116 million people in the United States. Current pain medications such as NSAIDS, steroids, opiates and gabapentin analogs have documented and often severe side effects, are poorly effective in neuropathic pain and provide adequate relief only in limited subsets of patients. Because of its high prevalence and poor treatment options, chronic pain results in socioeconomic costs estimated at $560-635 billion annually in the US. Activation of NMDA receptors (NMDARs) mediates central nervous system sensitization, which is implicated in the development and maintenance of neuropathic pain. NMDA-mediated central sensitization depends on formation of a multi-protein cascade complex at the receptor that includes post-synaptic density 95 protein (PSD95), neuronal nitric oxide synthase (nNOS) and NOS1 adaptor protein (NOS1AP). A peptide disruptor of the NMDAR multi-protein complex is efficacious in preclinical stroke and pain models and is currently in clinical trials for ischemic stroke. Small molecule inhibitors targeting this complex have the potential to be effective analgesics without the side effects associated with broad inhibition of NMDARs. A direct downstream effector of the NMDAR complex is nNOS-NOS1AP. A compound inhibiting this complex will likely be efficacious against neuropathic pain, stroke and chronic neurological diseases precipitated or exacerbated by excessive NMDAR activity. In the funded Phase I SBIR program, our team ran an extensive small molecule high-throughput screen to identify inhibitors of the nNOS-NOS1AP protein-protein interaction. After confirmation of activity, selectivity and initial administration-distribution-metabolism-excretion/toxicity (ADME/T) studies on the top leads, we chose two drug-like, selective nNOS-NOS1AP inhibitors with distinct scaffolds for in vivo studies. Both inhibitors are efficacious in pain models. We initiated a small chemistry effort on one chemical series, identifying regions for selectivity and potency. In the current proposal, a traditional drug medicinal chemistry approach will be used to design and develop novel analogs with improved pharmacokinetic properties and potency compared to the parent compounds. Anagin and its research partners at AMRI and Indiana University will advance at least one series through early lead optimization studies. In addition to improving potency and ADME/T properties, we will demonstrate that the best analogs are acting on the intended target in cells, validate their activity in two preclinical pain models and assess their safety profile in key behavioral in vivo models. Compounds that do not meet our set criteria will not be advanced. We anticipate that our lead compound would have a better therapeutic index than current pain medications. We have a team of business, chemistry, biology and in vivo scientific experts in place to advance these series towards lead optimization and into the clinic for the treatment of chronic neuropathic pain.